Bile acid salts

ABSTRACT

A process for the preparation of the compounds of general formula (I)  
                 
 
     in which  
     R 1  is H or OH;  
     R 2  is H, α-OH, or β-OH; and  
     R 3  is a straight or branched C 1 -C 4  alkyl group or a benzyl group,  
     comprising the reduction of compounds of formula (III)  
                 
 
     wherein R 1 , R 2  and R 3  have the same meanings as in formula I, in the presence of sodium borohydride.

[0001] The present invention relates to a novel process for thepreparation of bile acid derivatives in which an amino group is presentat the 3β position.

[0002] The most important bile acids are represented in the followingscheme 1:

[0003] More particularly, the invention relates to a process for thepreparation of compounds of formula (I) from compounds of formula (II):

[0004] in which

[0005] R₁ is H or OH;

[0006] R₂ is H, α-OH, or β-OH; and

[0007] R₃ is a straight or branched C₁-C₄ alkyl group, or a benzylgroup.

[0008] The compounds of general formula (I) are important intermediatesfor the preparation of compounds useful for a number of pharmaceuticalapplications. They are, for example, used for the preparation ofinhibitors of bile acids intestinal absorption (see EP-A-0489423) or ascarriers for active compounds in the enter-hepatic circulation (seeEP-A-0417725).

[0009] Last, but not least, is the use thereof for the preparation ofcontrast agents for medical diagnosis using Magnetic Resonance, such asthose described in WO-A-95/32741, resulting from the conjugation of abile acid with a chelating agent, which are capable of chelating theions of paramagnetic bi- and trivalent metals, in particular thegadolinium ion, or in the publication: Anelli P. L. et al., ActaRadiologica, 38, 125, 1997.

[0010] EP-A-614,908 discloses the preparation of the derivatives ofgeneral formula (I), comprising the following steps:

[0011] a) formation of phthalimido derivatives of general formula (III)by reacting compounds of general formula (II) with phthalimide;

[0012] b) treatment of compounds of general formula (III) with hydrazinehydrate or phenylhydrazine;

[0013] c) subsequent treatment of the reaction products from step b)with mineral acids to form the addition salts, and

[0014] d) liberation from the salt to give the compounds of generalformula (I).

[0015] As already widely discussed in EP-A-614,908, the formation of thephthalimido derivative is carried out by means of the well knownMitsunobu reaction (see Synthesis, 1, 1981; Org. React. Vol. 42, 335(1992)), namely in the presence of a suitable phosphine and DEAD(diethylazodicarboxylate) or DIAD (diisopropylazodicarboxylate), in anorganic solvent such as dioxane or tetrahydrofuran, at a temperaturewhich ranges from 20 to 50° C. The Mitsunobu reaction yields the finalproducts with inversion of configuration.

[0016] The process illustrated makes use of hydrazine hydrate orphenylhydrazine, particularly dangerous products due to theirascertained cancerogenicity.

[0017] It has now surprisingly been found that the reduction of thephthalimido group in the compounds of formula (III) can beadvantageously carried out with sodium borohydride.

[0018] It is therefore the object of the present invention a process forthe preparation of the compounds of general formula (I) comprising thereduction reaction of the compounds of general formula (III) in thepresence of sodium borohydride, according to the following Scheme 2:

[0019] in which

[0020] R₁ is H or OH;

[0021] R₂ is H, α-OH, or β-OH; and

[0022] R₃ is a straight or branched C₁-C₄ alkyl group, or a benzylgroup.

[0023] A further object of the present invention is the process for thepreparation of the compounds of general formula (I) comprising thereduction of the compounds of general formula (III) in the presence ofsodium borohydride, through formation of the novel compounds of generalformula (IV), and subsequent deprotection by treating said compoundswith acids, according to the following Scheme 3:

[0024] in which R₁, R₂ and R₃ have the meanings defined above.

[0025] Compounds of general formula (IV) are novel, useful intermediatesin the process according to Scheme 3 and their recovery will bedescribed in the Experimental Section.

[0026] Particularly preferred is the process for the preparation ofcompounds of formula (Ia), according to Scheme 3, starting fromcompounds of formula (IIIa), which are derivatives of cholic ordeoxycholic acid,

[0027] in which

[0028] R₃ has the meanings defined above and

[0029] R₄ can be a hydrogen atom or a hydroxy group.

[0030] A further object of the present invention is the process for thepreparation of compounds of formula (Ib), according to Scheme 4,starting from compounds of formula (IIIb), which are deoxycholic acidderivatives,

[0031] in which R₃ has the meanings defined above.

[0032] Particularly preferred is the process for the preparation ofcompound (Ic), according to Scheme 5, starting from compound (IIIc), adeoxycholic acid derivative,

[0033] The compounds of formula (IIIc) and (IVc),(3β,5β,12α)-3-[1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)]-12-hydroxycholan-24-oicacid methyl ester and(3β,5β,12α)-12-hydroxy-3-[[(2-hydroxymethyl)benzoyl]amino]cholan-24-oicacid methyl ester, respectively, are novel and they are usefulintermediates for the preparation of compound of formula (Ic), adeoxycholic acid derivative.

[0034] The compounds of formula (III) are synthesized according to thegeneral procedure already described above and exemplified inEP-A-614,908.

[0035] Preferred reaction conditions comprise:

[0036] 1) selection of a reaction temperature from 15 to 25° C., thusdecreasing the amount of diisopropylazodicarboxylate to a small excessto the stoichiometric;

[0037] 2) crystallization of phthalimido derivatives (III) from MeOHinstead of 2-PrOH, thereby reducing the volume of the crystallizationsolvent by at least four times.

[0038] The reduction of phthalimido derivatives with NaBH₄ has neverbefore been described in the field of the compounds of the presentinvention, but references exist in the literature, (T. W. Greene; P. G.M. Wuts “Protective groups in Organic Synthesis”; 3^(rd) Ed John Wileyand sons, New York, 1999), reporting the use of a large excess of thisreducing agent (5-10 mol) to deprotect a phthalimido group.

[0039] The teaching contained in the most important paper [Osby,Tetrahedron Letters, Vol. 25, 2093 (1984)] i.e. the use of2-PrOH/H₂O=6/1 as a reduction solvent proved to be definetly unsuitablefor the process of the present invention. In fact, due to the poorsolubility of compounds of formula (III), it was necessary to operate attemperatures around 40° C. and with a 2.5% maximum concentration whichis of course industrially unacceptable.

[0040] On the other hand, Osby already evidenced that the proposedconditions were not particularly suitable to deprotect phthalimidogroups from derivatives containing an ester function. Osby himself infact observed that the deprotection of this group in said derivativeswas accompanied by reduction and hydrolysis of the ester group withconsequent decrease in reaction yields.

[0041] The Applicant found that when reducing the compounds of formula(III) under the Osby conditions, compounds of formula (I) were recoveredin around 65% yields, but contaminated by remarkable amounts ofby-products, mainly the product from the reduction of the ester group at24- to alcohol. This phenomenon is still significant even when markedlyreducing the NaBH₄ excess. In order to better understand the genesis ofthe by-products, if the reaction carried out under the Osby conditionswas worked up at the end of the reduction with NaBH₄ and before the acidhydrolysis and the side-products recovered by silica chromatography hadthe formulae (V)-(VIII). These by-products are of course the precursorsof the impurities evidenced in compounds of formula (I) and account forthe low reaction yields.

[0042] In the Experimental section the recovery and the characterizationof the various intermediates, in particular in the case of deoxycholicacid, will be reported.

[0043] Compounds of formula (IV) are substantially inseparable fromcompounds (V) by crystallization from different solvents. Not even theconversion of compounds (IV) into the corresponding acids of formula(VI) allows the elimination of the by-products of formula (V).

[0044] It has surprisingly been found that using dipolar aproticsolvents such as DMA (dimethylacetamide), DMF (dimethylformamide), DMSO(dimethylsulfoxide), NMP (N-methyl-pyrrolidone), in place of analcoholic solvent, such as isopropanol described by Osby, these problemscould be overcome. Particularly preferred are dimethylacetamide andN-methylpyrrolidone.

[0045] Using said solvents the concentration of compounds (III) could beincreased up to more than 10%, without increasing the amount ofside-products.

[0046] Moreover, under the conditions of the present invention, theNaBH₄ excess could be decreased, compared with what reported above.

[0047] In particular, the molar equivalents of reducing agent can belowered from a strong excess (about 5) to substantially stoichiometricvalues (1.2-0.85) to the substrate, without adversely affecting thereaction yields.

[0048] A further aspect of the present invention is the use,simultaneously with the dipolar aprotic solvents, of a buffer solutionat pH 7.5-9 which allows to control pH during the reaction. This allowsto inhibit the hydrolysis of sensitive functions minimizing theformation of by-products (VI)-(VIII).

[0049] The compounds of formula (IV) are then transformed into those offormula (I) by treatment in dipolar aprotic or alcoholic solvent,optionally in mixture with water, and in the presence of a mineral ororganic acid (such as acetic acid).

[0050] Particularly preferred is the use of HCl in methanol solution.The subsequent liberation of the free base from the salt is carried outby treatment of the solution in one of the above cited solvents withaqueous bases (such as NaOH, Na₂CO₃ . . . ).

[0051] As already cited above, compounds of formula (I) are useful forthe preparation of medicaments for decreasing cholesterol plasmaticlevels, (EP-A-417,725 or EP-A-489,423) or of contrast agents for thenuclear magnetic resonance diagnosis, as described in WO-A-95/32741.

[0052] For the latter, the known synthesis comprised the transformationof compounds (II) into the corresponding compounds (I) by intermediateformation of the azide at the 3β position, according to Mitsunobureaction, as represented in the following Scheme 6:

[0053] This Synthetic scheme is unsuitable for scaling-up, being azidespotentially dangerous and the key reagent (diphenylphosphoryl azide)extremely expensive.

[0054] The present invention relates to a process for the preparation ofchelating agents of general formula (IX), capable of chelatingparamagnetic bi-trivalent metal ions, selected the group consisting ofFe⁽²⁺⁾, Fe⁽³⁺⁾, Cu⁽²⁺⁾, Cr⁽³⁺⁾, Gd⁽³⁺⁾, Eu⁽³⁺⁾, Dy⁽³⁺⁾, La⁽³⁺⁾, Yb⁽³⁺⁾or Mn⁽²⁺⁾,

[0055] in which

[0056] R₁, and R₂ have the meanings defined above;

[0057] R₅ is a hydrogen atom or a (C₁-C₅) alkyl group unsubstituted orsubstituted with a carboxylic group;

[0058] X is the residue of a polyaminocarboxylic ligand and ofderivatives thereof, selected from the group consisting of:ethylenediaminotetraacetic acid (EDTA), diethylenetriaminopentaaceticacid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) , 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),[10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid (HPDO3A),4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oicacid (BOPTA);

[0059] L is a residue of formula

[0060] m is an integer from 1 to 10, wherein for values above 1, thevalues of p and q can differ in the monomeric units;

[0061] q is 0 or 1;

[0062] p can range from 0 to 10, p and q not being at the same timezero,

[0063] said process comprising the following steps:

[0064] a) formation of compounds (III) starting from compounds (II) byreaction with phthalimide according to Mitsunobu's procedure, attemperatures ranging from 15 to 25° C., in the presence of anazodicarboxylate selected from DEAD (diethylazodicarboxylate) or DIAD(diisopropylazodicarboxylate) in amounts ranging from 1.1 to 1.3 molarequivalents, in a solvent selected from the group consisting of THF,dioxane, toluene and DMF;

[0065] b) reduction of compounds (III) with NaBH₄ to give compounds(IV);

[0066] c) acidic hydrolysis of compounds (IV) followed by neutralizationto give compounds (I);

[0067] d) condensation of compounds (I) with the reactive residues ofthe polyaminocarboxylic ligands defined above.

[0068] Particularly preferred is the process for the preparation ofcompounds (IXa), which are cholic or deoxycholic acid derivatives,according to the following Scheme 7:

[0069] in which R₃, R₄ and R₅ have the meanings defined above.

[0070] Furthermore, particularly preferred is the process for thepreparation of compounds (X), in which in formula (IXa) the residue X isDTPA substituted on the chain at the central position, R₆ can be ahydrogen atom or a carboxylic group and the L chain, R₄ and R₅ have themeanings defined above.

[0071] Particularly preferred is the process for the preparation ofcompounds (Xa)

[0072] in which the L chain, R₄ and R₅ have the meanings defined above.

[0073] Furthermore, particularly preferred is the process for thepreparation of the following novel compound, of general formula (Xa):

[0074][3β(S),5β,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid;

[0075] Other compounds belonging to this class, whose preparation hasalready been described in WO-A-95/32741, are the following:

[0076][3β(S),5β,7α,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0077][3β(S),5β,7α,12α]-3-[[4-[[5-[bis[2-[bis(carboxymethyl)-amino]-ethyl]amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]-amino]-7,12-dihydroxycholan-24-oicacid.

[0078] It is moreover preferred the process for the preparation of thefollowing compounds of formula (Xb), in which in formula (X) R₆ is ahydrogen atom, and R₄, R₅ and L have the meanings defined above.

[0079] Also preferred is the process for the preparation of thefollowing novel compound, belonging to the class of general formula(Xb):

[0080](3β,5β,7α,12α)-3-[[[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid

[0081] Other compounds belonging to this class, already described inWO-A-95/32741, are the following:

[0082](3β,5β,7α,12α)-3-[[[[[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0083](3β,5β,7α,12α)-3-[[6-[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]-1-oxohexyl]amino]-7,12-dihydroxycholan-24-oicacid.

[0084] Particularly preferred is also the process for the preparation ofcompounds of formula (XI), in which in formula (IXa) the residue Xderives from DTPA, and R₄, R₅ and L have the meanings defined above.

[0085] Other compounds belonging to this class, whose preparation wasalready described in WO-A-95/32741, are the following:

[0086] (3β,5β,7α,12α)-3-[[N-[N-[2-[[2-[bis(carboxymethyl)-amino]ethyl]-(carboxymethyl)amino]ethyl]-N-(carboxymethyl)-glycyl]glycyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0087]18-[[(3β,5β,7α,12α)-23-carboxy-7,12-dihydroxy-24-norcholan-3-yl]amino]-3,6,9-tris(carboxymethyl)-11,18-dioxo-3,6,9,12-tetraazaoctadecanoicacid.

[0088] Particularly preferred is also the process for the preparation ofthe following compounds of formula (XII), in which in formula (IXa) theresidue X is DOTA, and R₄, R₅ and L have the meanings defined above.

[0089] Furthermore, preferred is the process for the preparation ofcompounds of formula (XIII), in which in formula (IXa) the residue X isEDTA, and R₄, R₅ and L have the meanings defined above.

[0090] Particularly preferred is the process for the preparation of thefollowing compounds of formula (XIII):

[0091][3β(S),5β,7α,12α]-3-[[4-[[5-[[2-[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]amino]-7,12-dihydoxycholan-24-oicacid

[0092][3β(S),5β,12α]-3-[[4-[[2-[[bis(carboxymethyl)amino]-ethyl](carboxymethyl)amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid

[0093][3β(S),5β]-3-[[4-[[2-[[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]-4-carboxy-1-oxobutyl]amino]-12-oxocholan-24-oicacid

[0094] The experimental conditions used will be illustrated in detail inthe Experimental Section.

EXPERIMENTAL SECTION EXAMPLE 1(3β,5β,12α)-3-[1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)]-12-hydroxycholan-24-oicacid methyl ester

[0095]

[0096] Phthalimide (202.8 g; 1.378 mol) is suspended in a solution of(3β,5β,12α)-3,12-dihydroxycolan-24-oic acid methyl ester (prepared asdescribed in Steroids, vol. 37, 239) (511.9 g; 1.259 mol), andtriphenylphosphine (372.7 g; 1.421 mol) in THF (1.5 L), then a solutionof diisopropylazodicarboxylate (284.7 g; 1.408 mol) in THF (0.49 L) isdropped therein in 1.5 h, keeping the temperature at 15° C. Theresulting solution is left at room temperature for 18 h. The solvent isdistilled off and the oily residue is added with MeOH (3.2 L) andstirred for 20 h. The resulting crystalline product is filtered, washedwith MeOH (1.4 L) and dried to give the desired product (481.3 g; 0.898mol). Mother liquors and washings are concentrated to give a second cropof product (61.6 g; 0.114 mol) Yield: 80% m.p.: 160-162° C. HPLC assay:98.7% Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mmMerck KGaA; Temperature: 45° C.; Mobile phase: gradient elution A =0.017M H₃PO₄ in water B = CH₃CN Gradient: mm % A % B  0 82 18 30 15 8545 15 85 Flow: 1 mL min⁻¹; Detection (UV): 210 nm; Elemental analysis CH N % calc.: 73.99 8.47 2.61 % found: 73.96 8.51 2.62 TLC: silica gelplate 60F 254 Merck Eluent: AcOEt/n-hexane = 4:6 Rf: 0.35 Detection: 2%Ce(SO₄).4H₂O, 4.2% (NH₄)₆Mo₇O₂₄, 6% H₂SO₄ in water

[0097] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

EXAMPLE 2

[0098] Reduction of compounds of formula (III) according to a proceduresimilar to that described in Osby, Tetrahedron Letters, Vol. 25, 2093(1984).

[0099] (3β,5β,12α,)-3-Amino-12-hydroxycholan-24-oic acid methyl ester

[0100] The compound prepared in Example 1 (75 g; 0.14 mol) is dissolvedin 2-PrOH (2.5 L) at 75° C. The solution is vigorously stirred andcooled to 50° C., then added with H₂O (430 mL) to obtain a suspensionwhich is added with solid NaBH₄ (26.5 g; 0.70 mol) in portions during 30min. The reaction mixture is stirred at 40° C. for 2.5 h. CH₃COOH iscarefully added to pH 4.5-5 and the mixture is heated at 80° C. for 24h, keeping pH 4.5-5 by repeated additions of CH₃COOH. After cooling toroom temperature and evaporation of the solvent, a residue is obtainedwhich is dissolved in water (1 L). The solution is alkalinized to pH 10with 2M NaOH to obtain a precipitate which is stirred for 1 h, thenfiltered, washed with water and dried.

[0101] Crystallization from acetonitrile (1.2 L) yields the desiredproduct (36.9 g; 0.091 mol) Yield: 65% HPLC assay: 87% Stationary phase:Lichrosorb RP-Select B 5 μm; column 250 × 4 mm Merck KGaA; Temperature:45° C.; Mobile phase: gradient elution A = 0.01M sodium pentanesulfonatein water buffered to pH 2.5 with H₂SO₄ B = CH₃CN Gradient: mm % A % B  082 18 30 15 85 45 15 85 Flow: 1 mL min⁻¹; Detection (UV) 210 nm;

[0102] Silica gel chromatography of the crude compound gave the mainby-product (2.5 g) which was:

m.p.: 174-175° C. Elemental analysis C H N % calc.: 76.34 11.48 3.71 %found: 76.18 11.65 3.52

[0103] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

[0104] In another reaction carried out under the above describedconditions, at the end of the reduction the NaBH₄ excess was destroyedwith acetic acid and the reaction mixture (about neutral pH) wasevaporated and taken up with water to obtain a crude which was purifiedby silica gel chromatography. Together with the expected compound (IVb)(see EXAMPLE 3) remarkable amounts of the by-products, reportedhereinbelow together with their analytical characterization, wererecovered. This widely accounts for the low recovery yield of compound(Ib) after hydrolysis with acetic acid at 80° C.

[0105]N-[(3β,5β,12α)-12,24-Dihydroxycholan-3-yl]-2-(hydroxymethyl)benzamide

m.p.: 200-203° C. Elemental analysis C H N % calc.: 75.11 9.65 2.74 %found: 74.66 9.61 2.71

[0106](3β,5β,12α)-12-Hydroxy-3-[[2-(hydroxymethyl)benzoyl]-amino]cholan-24-oicacid

m.p.: >260° C. Elemental analysis C H N % calc.: 73.11 9.01 2.66 %found: 73.32 9.15 2.76

[0107]N-[(3β,5β,12α)-12-Hydroxy-24-methoxy-24-oxocholan-3-yl]-2-(hydroxymethyl)benzamide

[0108] The product was characterized as ammonium salt m.p.: 156-162° C.Elemental analysis C H N % calc. 69.44 8.83 4.91 % found: 69.27 8.894.82

[0109] (3β,5β,12α)-12-Hydroxy-3-[[2-carboxybenzoyl]amino]-cholan-24-oicacid

m.p. 193-196° C. Elemental analysis C H N % calc.: 71.48 8.06 2.60 %found: 71.18 8.25 2.55

[0110] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

EXAMPLE 3(3β,5β,12α,)-12-Hydroxy-3-[[(2-hydroxymethyl)benzoyl]-amino]cholan-24-oicacid methyl ester

[0111]

[0112] A solution of the compound prepared in Example 1 (225 g; 0.42mol) in DMA (1.75 L), vigorously stirred at 34° C., is diluted with 2Mbuffer phosphate pH 8 (500 mL) obtaining a mixture at 43° C. Solid NaBH₄(15.45 g; 0.408 mol) is added in 5 min and the suspension is stirred at47-48° C. After 55 min an almost clear solution is obtained which isadded with CH₃COOH to neutralize the hydride excess and adjust pH from12.8 to about 8.

[0113] The mixture is poured into H₂O and stirred for 3 h, theprecipitate is filtered and suspended in H₂O then stirred for 30 min.Finally, the precipitate is filtered, washed with H₂O and dried to givethe desired product (224.6 g; 0.416 mol) Yield: 99% m.p.: 190.2-192.7°C. HPLC assay: 93% Stationary phase: Lichrosorb RP-Select B 5 μm; column250 × 4 mm Merck KGaA; Temperature: 45° C.; Mobile phase: gradientelution A = 0.017M H₃PO₄ in water B = CH₃CN Gradient: mm % A % B  0 8218 30 15 85 45 15 85 Flow: 1 mL min⁻¹; Detection (UV): 210 nm; Elementalanalysis C H N % calc.: 73.43 9.15 2.60 % found: 73.43 9.19 2.56 TLC:silica gel plate 60F 254 Merck Eluent: AcOEt/n-hexane/MeOH = 5:5:1 Rf:0.79 Detection: 2% Ce(SO₄).4H₂O, 4.2% (NH₄)₆Mo₇O₂₄, 6% H₂SO₄ in water

[0114] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

EXAMPLE 4 (3β,5β,12α,)-3-Amino-12-hydroxycholan-24-oic acid methyl esterhydrochloride

[0115]

[0116] A suspension of the compound prepared in Example 3 (223.5 g;0.414 mol) in MeOH (1.5 L) and 37% HCl (52 mL; 0.63 mol) is heated at50° C. under stirring. After about 1.5 h a solution is obtained andafter a further 2 h, the reaction is completed. The solvent isevaporated off and the residue is stirred at 60° C. for 2 h with CH₃CN.After 15 h at room temperature the precipitate is filtered, washed withCH₃CN and dried to give the desired product (183.3 g; 0.414 mol). Yield:100% m.p.: >250° C. HPLC assay: 95% Stationary phase: LichrosorbRP-Select B 5 μm; column 250 × 4 mm Merck KGaA; Temperature: 45° C.;Mobile phase: gradient elution A = 0.01M sodium pentanesulfonate inwater buffered to pH 2.5 with H₂SO₄ B = CH₃CN Gradient: mm % A % B  0 8218 30 15 85 45 15 85 Flow: 1 mL min⁻¹; Detection (UV): 210 nm;

EXAMPLE 5 (3β,5β,12α,)-3-Amino-12-hydroxycholan-24-oic acid methyl ester

[0117]

[0118] The compound prepared in Example 4 (89.5 g; 0.202 mol) isdissolved in MeOH (460 mL) at 55° C., then the solution is cooled toabout 40° C. and poured, under strong stirring, into H₂O while addingsolid Na₂CO₃ to keep pH 9.8. After stirring for 2 h, the suspension isfiltered, the solid is suspended in H₂O and stirred for 2 h, filteredand washed with H₂O. After drying, the desired product is obtained (74.9g; 0.185 mol). Yield: 91% m.p.: 155-155.5° C. HPLC assay: 96.5%Stationary phase: Lichrosorb RP-Select B 5 μm; column 250 × 4 mm MerckKGaA; Temperature: 45° C.; Mobile phase: gradient elution A = 0.01Msodium pentanesulfonate in water buffered to pH 2.5 with H₂SO₄ B = CH₃CNGradient: mm % A % B  0 82 18 30 15 85 45 15 85 Flow: 1 mL min⁻¹;Detection (UV): 210 nm; GC assay: 96.3% Stationary phase: PE 1 (OV 1)Film thickness: 0.25 μm Column (WCOT): 30 m × 0.32 mm Startingtemperature 255° C., after 5 min the in the oven: temperature isincreased by 0.6° C./min to reach 290° C. Injector 290° C. temperature:Temperature FID: 290° C. Elemental analysis C H N % calc.: 74.03 10.693.45 % found: 73.91 10.78 3.44 TLC: silica gel plate 60F 254 MerckEluent: CHCl₃/MeOH/25% NH₄OH = 6:2:0.5 Rf: 0.69 Detection: 2%Ce(SO₄).4H₂O, 4.2% (NH₄)₆Mo₇O₂₄, 6% H₂SO₄ in water

[0119] The ¹H-NMR, 13C-NMR, IR and MS spectra are consistent with theindicated structure.

[0120] Compounds of formula (I), whose formulae and analytical data arereported hereinbelow, were prepared analogously: m.p. (° C.) Rf^(a)

164-166 0.48

121-123 0.67

127-128 0.80

[0121] The ¹H-NMR, ¹³C-NMR, IR and MS spectra as well as the elementaryanalysis are consistent with the indicated structure.

EXAMPLE 6[3β(S),5β,12α]-3-[[4-[Bis[2-[bis(carboxymethyl)amino]-ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid

[0122]

[0123] A)[3β(S),5β,12α]-3-[[4-[Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]amino]-5-(1,1-dimethylethoxy)-1,5-dioxopentyl]amino]-12-hydroxycholan-24-oicacid methyl ester

[0124] Triethylamine (2.23 g; 22 mmol) is added to a solution of 8.93 gof [3β,5β,12α]-3-amino-12-hydroxycholan-24-oic acid methyl ester(prepared in Example 5) (22 mmol), 16.41 g of N,N-bis[2- [bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid(1,1-dimethylethyl) ester (prepared as described in WO-A-95/32741:example 15) (22 mmol) and 3.91 g of diethyl cyanophosphate (24 mmol) in300 mL of DMF at 0° C. After 1.5 h at 0° C. and 18 h at roomtemperature, the reaction mixture is evaporated and the residue isdissolved in AcOEt. The solution is washed with a NaHCO₃ saturatedsolution and H₂O, dried (Na₂SO₄) and evaporated. The crude is purifiedby flash chromatography to give the desired product (20.67 g; 18.2mmol). Yield: 83% [α]²⁰ _(D) = −6.75 (c 2.0, CHCl₃) Elemental analysis CH N % calc. 65.69 9.60 4.94 % found: 66.54 9.95 4.99 TLC: Carrier:silica gel plate 60F 254 Merck Eluent: n-hexane/EtOAc = 1:1 R_(f) = 0.09Detection: Ce(SO₄)₂.4H₂O (0.18%) and (NH₄)₆Mo₇O₂₄.4H₂O (3.83%) in 10%H₂SO₄

[0125] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

[0126] B)[3β(S),5β,12α]-3-[[4-[Bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid

[0127] The compound prepared at step A) (19.72 g; 17.4 mmol) isdissolved in 105 mL of CF₃CO₂H at room temperature. After 26 h thesolution is evaporated and the residue is treated with H₂O; the solid isfiltered, washed with H₂O and partially dried under vacuum. Theresulting intermediate is dissolved in H₂O at pH 13 with 1M NaOH.

[0128] After 5 h at room temperature the solution is dropwise added with0.5M HCl at pH 1.4. After 15 h at room temperature the precipitate isfiltered, washed with H₂O and dried under vacuum to give a crude whichis purified by chromatography on Amberlite® XAD 1600 resin to obtain thedesired product (9.92 g; 11.8 mmol). Yield: 68% m.p. 184° C. (dec.)Compleximetric titer 99.3% (0.1M GdCl₃): Acidic titer 99.8% (0.1M NaOH):[α]²⁰ _(D) = (c 2.0; 1M NaOH) λ(nm) 589 578 546 436 405 365 [α] +23.61+24.59 +27.90 +46.67 +55.61 +71.40 Elemental analysis C H N % calc.:58.70 7.93 6.68 % found: 58.22 8.16 6.59 H₂O 0.70% TLC: silica gel plate60F 254 Merck Eluent: CHCl₃/MeOH/NH₄OH = 5:4:2 R_(f) = 0.13 Detection:Ce(SO₄)₂.4H₂O (0.18%) and (NH₄)₆Mo₇O₂₄.4H₂O (3.83%) in 10% H₂SO₄

[0129] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

EXAMPLE 7(3β,5β,7α,12α)-3-[[[Bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid

[0130]

[0131] A)N-[[Bis[2-[bis[2-(1,1-dimethylethoxy-2-oxoethyl]-amino]ethyl]amino]-acetyl]glycine

[0132] 6.5 g of glycylglycine (49.3 mmol) are suspended in 100 mL of a1:1=H₂O:EtOH mixture and dissolved at pH 10 with 10M NaOH (4.8 mL).N-(2-bromoethyl)-N[2-(1,1-dimethylethoxy)-2-oxoethyl]glycine-1,1-dimethylethylester (42 g; 110.9 mmol) in 40 mL of EtOH is dropped in 2 h keeping pHat 10.5 with 10M NaOH (5.8 mL). The solution quickly turns to anemulsion, which is dissolved after 2.5 h by addition of 10M NaCH. After22 h the solvent is evaporated off, the residue is diluted with waterand extracted with CH₂Cl₂. The organic phase is washed with H₂O, driedand evaporated, to give a residue which is purified by flashchromatography. The residue is dissolved in water, pH is adjusted to 4.5by addition of 1M HCl and the solution is extracted with chloroform. Theorganic phase is washed with H₂O, dried and evaporated, to give 13 g ofthe desired compound (19.3 mmol). Yield: 39% Elemental analysis C H N %calc. 56.95 8.66 8.30 % found: 56.67 8.68 8.30 TLC: silica gel plate 60F254 Merck Eluent: CHCl₃/MeOH/NH₄OH 25% = 6:3:1 R_(f) = 0.65 Detection:KMnO₄ in alkaline solution

[0133] The ¹H-NMR, ¹³C-NMR,IR and MS spectra are consistent with theindicated structure.

[0134] B)(3β,5β,7α,12α)-3-[[[[[Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]amino]acetyl]amino]acetyl]-amino]-7,12-dihydroxycholan-24-oicacid methyl ester

[0135] 2.8 mL of TEA (20.2 mmol) are dropwise added in 5 min to asolution of 13.6 g of the compound prepared at step A) (20.2 mmol), 8.52g of (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid methylester (prepared in Example 5) (20.2 mmol) and DEPC (3.4 mL; 22.2 mmol)in DMF (290 ml) stirring at 0° C. After 1 h the reaction is warmed toroom temperature and the solution is stirred for 6.5 h. 0.3 mL of DEPC(2 mmol) are added and the solution is stirred for a further 15.5 h. DMFis evaporated off, the residue is dissolved in EtOAc, washed with aq.NaHCO₃ and then water and finally dried. Purification by flashchromatography yields 13.7 g of the desired product (12.7 mmol). Yield:63% [α]²⁰ _(D) = +5.26 (c 1.5; CHCl₃) Elemental analysis C H N % calc.63.48 9.25 6.49 % found: 63.22 9.40 6.40

[0136] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

[0137] C)(3β,5β,7α,12α)-3-[[[[[Bis[2-[bis(carboxymethyl)amino]ethyl]-amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid

[0138] 12.85 g of the compound prepared at step B) (12 mmol) aredissolved in TFA (210 mL) stirring at 0-5° C. After 16 h TFA isevaporated off to give a residue which is dissolved in 90 mL of 0.8MNaOH at pH 13 and stirred at room temperature for 15 h. The solution isconcentrated to 50 mL, dropped in 105 mL of 0.6M HCl and stirred for 2h. The solid is filtered, washed with 0.1M HCl and dried to obtain acrude which is purified by chromatography. The fractions containing thedesired compound in salified form are evaporated to give a residue whichis dissolved in water and dropped into 1M HCl at pH 1.45. Theprecipitate is filtered, washed with 0.1M HCl and dried to give 2.6 g ofthe desired product (3.1 mmol). Yield: 26% m.p.: 120-125° C. HPLC assay:98% (area %)

[0139] The ¹H-NMR, ¹³C-NMR, IR and MS spectra are consistent with theindicated structure.

EXAMPLE 8

[0140] Preparation of cholic acid derivative chelating agents.

[0141] Using (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acidmethyl ester prepared as described in Example 5, and following theprocedures described in WO-A-95/32741, the following chelating agentswere prepared:

[0142][3β(S),5β,7α,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0143][3β(S),5β,7α,12α]-3-[[4-[[5-[bis[2-[bis(carboxymethyl)-amino]-ethyl]amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]-amino]-7,12-dihydroxycholan-24-oicacid;

[0144](3β,5β,7α,12α)-3-[[[[[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0145](3β,5β,7α,12α)-3-[[6-[[[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetyl]amino]-1-oxohexyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0146] (3β,5β,7α,12α)-3-[[N-[N-[2-[[2-[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]ethyl]-N-(carboxymethyl)glycyl]glycyl]amino]-7,12-dihydroxycholan-24-oicacid;

[0147]18-[[(3β,5β,7α,12α)-23-carboxy-7,12-hydroxy-24-norcholan-3-yl]amino]-3,6,9-tris(carboxymethyl)-11,18-dioxo-3,6,9,12-tetraazaoctadecanoicacid.

1. A process for the preparation of compounds of general formula (I)

in which R₁ is H or OH; R₂ is H, α-OH, or β-OH; and R₃ is a straight orbranched C₁-C₄ alkyl group, or a benzyl group; comprising the reductionof compounds of general formula (III)

 wherein R₁, R₂ and R₃ have the same meanings as in formula (I), in thepresence of sodium borohydride.
 2. A process as claimed in claim 1,which comprises the preparation of compounds of general formula (IV),

in which R₁, R₂ and R₃ have the same meanings as in claim 1, and thesubsequent deprotection by acid treatment.
 3. A process as claimed inclaim 2, for the preparation of compounds of general formula (Ia),

in which R₃ has the same meanings as in claim 1 and R₄ can be a hydrogenatom or a hydroxyl group.
 4. A process as claimed in claim 3, for thepreparation of compounds of general formula (Ib),

in which R₃ has the same meanings as in claim
 1. 5. A process as claimedin claim 4, for the preparation of the compound of formula (Ic)


6. A process according to claims 1 to 5, in which the reduction withsodium borohydride is carried out in a dipolar aprotic solvent selectedfrom the group consisting of DMA (dimethylacetamide), DMF(dimethylformamide), DMSO (dimethylsulfoxide), NMP(N-methylpyrrolidone).
 7. A process as claimed in claim 6, in which thereduction with sodium borohydride is carried out in a dipolar aproticsolvent selected from DMA and NMP.
 8. A process according to claims 1 to7, in which NaBH₄ mols range from 1.2 to 0.85.
 9. A process according toclaims 1 to 8, in which the reduction with sodium borohydride is carriedout in a dipolar aprotic solvent and in the presence of a buffersolution at pH 7.5-9.
 10. A process according to claims 1 to 9, in whichcompounds of formula (IV) are transformed into those of formula (I) bytreatment in a dipolar aprotic solvent, optionally in mixture withwater, and in the presence of a mineral or organic acid.
 11. A processas claimed in claim 10, in which the mineral acid is HCl in methanolsolution. 12.(3β,5β,12α)-3-[1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)]-12-hydroxycholan-24-oicacid methyl ester of formula (IIIc)


13. Compounds of general formula (IV)

in which: R₁ is H or OH; R₂ is H, α-OH, or β-OH; and R₃ is a straight orbranched C₁-C₄ alkyl group or a benzyl group.
 14. As a compound asclaimed in claim 7,(3β,5β,12α)-12-hydroxy-3-[[(2-hydroxymethyl)benzoyl]amino]cholan-24-oicacid methyl ester of formula (IVc)


15. A process for the preparation of the chelating agents of formula(IX), capable of chelating paramagnetic bi-trivalent metal ions selectedfrom the group consisting of Fe⁽²⁺⁾, Fe⁽³⁺⁾, Cu⁽²⁺⁾, Cr⁽³⁺⁾, Gd⁽³⁺⁾,Eu⁽³⁺⁾, Dy⁽³⁺⁾, La⁽³⁺⁾, Yb⁽³⁺⁾ or Mn⁽²⁺⁾,

in which R₁ is H or OH; R₂ is H, α-OH, or β-OH; and R₅ is a hydrogenatom or a (C₁-C₅) alkyl group unsubstituted or substituted with acarboxylic group; X is the residue of a polyaminocarboxylic ligand andof derivatives thereof, selected from the group consisting of:ethylenediaminotetraacetic acid (EDTA), diethylenetriaminopentaaceticacid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),[10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid (HPDO3A),4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oicacid (BOPTA); L is a residue of formula

m is an integer from 1 to 10, wherein for values above 1, the values ofp and q can differ in the monomeric units; q is 0 or 1; p can range from0 to 10, p and q being not at the same time zero, said processcomprising the following steps:

a) preparation of compounds of formula (III) starting from compounds offormula (II) by reaction with phthalimide according to Mitsunobu'sprocedure, at temperatures ranging from 15 to 25° C., in the presence ofan azodicarboxylate selected from DEAD (diethylazodicarboxylate) or DIAD(diisopropylazodicarboxylate) in amounts from 1.1 to 1.3 molarequivalents, in a solvent selected from the group consisting of THF,dioxane, toluene and DMF; b) reduction of compounds of formula (III)with NaBH₄ to give compounds of formula (IV); c) acid hydrolysis ofcompounds of formula (IV) followed by neutralization to give compoundsof formula (I); d) condensation of compounds of formula (I) withreactive residues of the polyaminocarboxylic ligands defined above. 16.A process as claimed in claim 15 for the preparation of compounds offormula (IXa),

wherein R₄, R₅, X and L have the same meanings as in claim
 14. 17. Aprocess as claimed in claim 15 for the preparation of compounds offormula (IXb),

in which X, L and R₅ have the same meanings as in claim
 15. 18. Aprocess as claimed in claim 15 for the preparation of compounds offormula (X),

in which R₆ can be a hydrogen atom or a carboxylic group and L, R₄ andR₅ have the same meanings as in claim
 14. 19. A process as claimed inclaim 18 for the preparation of compounds of formula (Xa)

in which L, R₄ and R₅ have the same meanings as in claim
 13. 20. Aprocess as claimed in claim 19 for the preparation of the followingcompounds:[3β(S),5β,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid;[3β(S),5β,7α,12α]-3-[[4-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;[3β(S),5β,7α,12α]-3-[[4-[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]-amino]-7,12-dihydroxycholan-24-oicacid.
 21. A process as claimed in claim 15 for the preparation ofcompounds of formula (Xb),

in which R₄, R₅ and L have the same meanings as in claim
 15. 22. Aprocess as claimed in claim 21 for the preparation of the followingcompounds:(3β,5β,7α,12α)-3-[[[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid;(3β,5β,7α,12α)-3-[[[[[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]acetyl]amino]acetyl]amino]-7,12-dihydroxycholan-24-oicacid;(3β,5β,7α,12α)-3-[[6-[[[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetyl]amino]-1-oxohexyl]amino]-7,12-dihydroxycholan-24-oicacid.
 23. A process as claimed in claim 15 for the preparation ofcompounds of formula (XI),

in which R₄, R₅ and L have the same meanings as in claim
 15. 24. Aprocess as claimed in claim 23 for the preparation of the followingcompounds:(3β,5β,7α,12α)-3-[[N-[N-[2-[[2-[bis(carboxymethyl)amino]-ethyl](carboxymethyl)amino]ethyl]-N-(carboxymethyl)glycyl]-glycyl]amino]-7,12-dihydroxycholan-24-oicacid;18-[[(3β,5β,7α,12α)-23-carboxy-7,12-dihydroxy-24-norcholan-3-yl]amino]-3,6,9-tris(carboxymethyl)-11,18-dioxo-3,6,9,12-tetraazaoctadecanoicacid.
 25. A process as claimed in claim 15 for the preparation ofcompounds of formula (XII),

in which R₄, R₅ and L have the same meanings as in claim
 15. 26. Aprocess as claimed in claim 15 for the preparation of compounds offormula (XIII),

in which R₄, R5 and L have the same meanings as in claim
 15. 27. Aprocess as claimed in claim 26 for the preparation of the followingcompounds:[3β(S),5β,7α,12α]-3-[[4-[[5-[[2-[bis(carboxymethyl)amino]-ethyl](carboxymethyl)amino]-5-carboxypentyl]amino]-1,4-dioxobutyl]amino]-7,12-dihydroxycholan-24-oicacid;[3β(S),5β,12α]-3-[[4-[[2-[[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]-4-carboxy-1-oxobutyl]amino]-12-hydroxycholan-24-oicacid;[3β(S),5β]-3-[[4-[[2-[[bis(carboxymethyl)amino]ethyl]-(carboxymethyl)amino]-4-carboxy-1-oxobutyl]amino]-12-oxocholan-24-oicacid.